CN109589324B

CN109589324B - Therapeutic agent for keratoconjunctival disorder

Info

Publication number: CN109589324B
Application number: CN201811479227.3A
Authority: CN
Inventors: 木村和博
Original assignee: Yamaguchi University NUC
Current assignee: Yamaguchi University NUC
Priority date: 2012-11-08
Filing date: 2013-11-07
Publication date: 2022-06-14
Anticipated expiration: 2033-11-07
Also published as: US20230255937A1; MX2015005842A; CL2018001915A1; US11471440B2; US9750721B2; US20200345695A1; US20190134002A1; CN105188753B; KR102173932B1; ES2834111T3; US9492431B2; CA2890424C; CN105188753A; SG11201503612QA; EP2918290A4; AU2018217197A1; RU2659203C2; RU2015121628A; JP2019069992A; US20170065562A1

Abstract

The present invention addresses the problem of providing novel therapeutic agents for keratoconjunctival disorders. As a means for solving this problem, a therapeutic agent for keratoconjunctival disorder containing an RAR γ agonist as an active ingredient is provided. The therapeutic agent shows an excellent ameliorating effect in a keratoconjunctival disorder model, and thus can be used as a therapeutic agent for a keratoconjunctival disorder such as corneal ulcer, corneal epithelial abrasion, keratitis, dry eye, conjunctivitis, chronic superficial keratitis, corneal erosion, persistent corneal disorders, superficial punctate keratopathy, corneal epithelial defects, conjunctival epithelial defects, keratoconjunctivitis sicca, superior limbic keratoconjunctivitis, filamentary keratoconjunctivitis, infectious keratitis, non-infectious keratitis, infectious conjunctivitis and non-infectious conjunctivitis. The therapeutic agent may also be used as a therapeutic agent for both corneal and conjunctival scarring associated with keratoconjunctival disorders.

Description

Therapeutic agent for keratoconjunctival disorder

The application is a divisional application of an invention patent application with application date of 2013, 11 and 7, application number of 201380069723.0 and named as a therapeutic agent for keratoconjunctival disorders.

Technical Field

The present invention relates to a therapeutic agent for keratoconjunctival disorders comprising an RAR gamma agonist as an active ingredient.

Background

The cornea is a transparent avascular tissue of about 1 cm diameter that covers the anterior surface of the eyeball. The conjunctiva is a mucous membrane covering the back of the eyelids and the surface of the eyeball, which is located behind the limbus. The cornea and conjunctiva play an important role in vision. It is known that when a disorder occurs therein, visual function is seriously impaired. Keratoconjunctival disorders induced by various diseases such as corneal ulcer, keratitis, and dry eye are disorders caused by: a delay in recovery from a condition caused by some cause, such as an external injury, or a condition that has become chronic. Because the cornea is a tissue that is connected to the conjunctiva, such diseases negatively affect each other in the normal composition of the epithelium and in some cases may cause damage to the structure or function of the corneal stroma or endothelium.

Collagen (particularly type I collagen) is known as one of the representative stromal components of corneal parenchymal tissue. Dysfunction due to matrix degradation occurs in diseases resulting from keratoconjunctival disorders. Therefore, inhibition of degradation of collagen (particularly type I collagen) is considered to be effective for diseases caused by keratoconjunctival disorders.

In keratoconjunctival disorders, scar tissue, which is often formed after inflammation has subsided, may interfere with visual function. For this reason, as in the inhibition of collagen degradation described above, inhibition of collagen contraction is considered to be effective, if possible, for contraction and formation of scars (hereinafter collectively referred to as "scar formation").

Patent document 1 describes that all-trans retinoic acid (hereinafter also referred to as ATRA) promotes corneal regeneration. However, its action is weak and its detailed mechanism has not been elucidated.

Further, ATRA is an agonist of retinoic acid receptor (hereinafter also referred to as RAR). However, since ATRA does not have selectivity over RAR subtypes RAR α, RAR β, and RAR γ, the contribution of each RAR subtype to corneal regeneration is unknown.

Meanwhile, RAR is involved in various roles such as growth, morphogenesis and differentiation in many cells such as inflammatory cells, immune cells and structural cells. Further, it was confirmed that there was a difference in the distribution of RAR subtypes depending on tissues or organs of mammals.

Some effects of RAR are undesirable, such as an increase in triglycerides by RAR α. Thus, specificity or selectivity relative to subtype in compounds with RAR agonist activity is expected to achieve a reduced risk of side effects.

For the above reasons, there is a need for RAR agonists that have a potent keratoconjunctival disorder-inhibiting effect and have high safety based on subtype selectivity.

Patent documents

2 and 3 disclose RAR agonist (E) -4- (2- {3- [ (1H-pyrazol-1-yl) methyl ] -5,5,8, 8-tetramethyl-5, 6,7, 8-tetrahydronaphthalen-2-yl } vinyl) benzoic acid and derivatives thereof. Further, patent document 2 describes that (E) -4- (2- {3- [ (1H-pyrazol-1-yl) methyl ] -5,5,8, 8-tetramethyl-5, 6,7, 8-tetrahydronaphthalen-2-yl } vinyl) benzoic acid is useful for emphysema, cancer, and skin diseases. Patent document 3 describes that the above agonist is useful for neuropathic pain.

In addition, non-patent document 1 describes that RAR agonist 6- [3- (1-adamantyl) -4-hydroxyphenyl ] -2-naphthoic acid induces apoptosis in lung cancer cells.

Further, patent document 4 describes that the RAR agonist 3-fluoro-4- [ 2-hydroxy-2- (5,5,8, 8-tetramethyl-5, 6,7, 8-tetrahydronaphthalen-2-yl) acetylamino ] benzoic acid is useful in muscle repair or regeneration.

However, for any RAR agonist, no pharmacological effect on keratoconjunctival disorders or scarring due to keratoconjunctival disorders is discussed or reported. In addition, there is no document suggesting such an effect.

Reference list

Patent literature

Patent document 1: japanese patent laid-open No. 2009 and 235031

Patent document 2: international publication No. WO 2002/028810

Patent document 3: international publication No. WO 2008/057930

Patent document 4: japanese patent laid-open No. 2013-5-536855

Non-patent document

Non-patent document 1: sun SY, Cancer Research 62 (8): 2430-.

Disclosure of Invention

Summary of The Invention

Technical problem

Finding effective drugs for ophthalmic diseases, especially keratoconjunctival disorders, is an important and interesting goal. It is an object of the present invention to provide a therapeutic agent which has an inhibitory effect on keratoconjunctival disorders and has high safety based on subtype selectivity.

Solution to the problem

In a diligent study seeking effective drugs for keratoconjunctival disorders, the present inventors found, through pharmacological testing using rabbit keratocytes and subconjunctival fibroblasts: the RAR γ agonist (E) -4- (2- {3- [ (1H-pyrazol-1-yl) methyl ] -5,5,8, 8-tetramethyl-5, 6,7, 8-tetrahydronaphthalen-2-yl } ethenyl) benzoic acid (R667: hereinafter also referred to as "RAR γ agonist a") exerts an excellent effect of ameliorating keratoconjunctival disorders and scarring associated with the keratoconjunctival disorders, in which a potent effect of inhibiting collagen degradation and a significant effect of inhibiting collagen contraction are demonstrated. In addition, other RAR γ agonists were found: 6- [3- (1-adamantyl) -4-hydroxyphenyl ] -2-naphthoic acid (CD 437: hereinafter also referred to as "RAR γ agonist B") and 3-fluoro-4- [ 2-hydroxy-2- (5,5,8, 8-tetramethyl-5, 6,7, 8-tetrahydronaphthalen-2-yl) acetylamino ] benzoic acid (BMS 961: hereinafter also referred to as "RAR γ agonist C") also showed a significant effect of inhibiting collagen degradation in pharmacological tests using rabbit corneal cells, thereby completing the present invention.

Specifically, the present invention is: [1] a therapeutic agent for keratoconjunctival disorder comprising an RARgamma agonist as an active ingredient, and [2] the therapeutic agent of the above [1], wherein the RARgamma agonist is (E) -4- (2- {3- [ (1H-pyrazol-1-yl) methyl ] -5,5,8, 8-tetramethyl-5, 6,7, 8-tetrahydronaphthalen-2-yl } vinyl) benzoic acid, 6- [3- (1-adamantyl) -4-hydroxyphenyl ] -2-naphthoic acid (naphalene acid), 3-fluoro-4- [ 2-hydroxy-2- (5,5,8, 8-tetramethyl-5, 6,7, 8-tetrahydronaphthalen-2-yl) acetylamino ] benzoic acid, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, An ester thereof or a salt thereof, [3] the therapeutic agent of the above [1] or [2], wherein the keratoconjunctival disorder is selected from the group consisting of corneal ulcer, corneal epithelial abrasion (corneal epithelial abrasion), keratitis, dry eye, conjunctivitis, chronic superficial keratitis, corneal erosion, persistent corneal disorder, superficial punctate keratopathy, corneal epithelial defect, conjunctival epithelial defect, keratoconjunctivitis sicca, superior limbal keratoconjunctivitis, filamentous keratoconjunctivitis, infectious keratitis, non-infectious keratitis, infectious conjunctivitis, non-infectious conjunctivitis, corneal scar and conjunctival scar, [4] the therapeutic agent according to any one of the above [1] to [3], wherein the administration form is instillation (intrasclatitive) administration or oral administration, and [5] the therapeutic agent according to any one of the above [1] to [4], wherein the dosage form is instillation, ophthalmic ointment, injection, eye drop, eye, Tablets, granules, fine granules, powders or capsules.

Advantageous effects of the invention

The RAR γ agonist as an active ingredient of the therapeutic agent for keratoconjunctival disorders of the present invention is useful as a therapeutic agent for keratoconjunctival disorders such as corneal ulcer, corneal epithelial abrasion, keratitis, dry eye, conjunctivitis, chronic superficial keratitis, corneal erosion, persistent corneal disorders, superficial punctate keratopathy, corneal epithelial defects, conjunctival epithelial defects, keratoconjunctivitis sicca, superior limbic keratoconjunctivitis, filamentous keratoconjunctivitis, infectious keratitis, non-infectious keratitis, infectious conjunctivitis, or non-infectious conjunctivitis, by strongly inhibiting the degradation of keratoconjunctival collagen.

Further, RAR γ agonists, which are effective ingredients of the therapeutic agents for keratoconjunctival disorders of the present invention, can also be used as therapeutic agents for corneal scars or conjunctival scars associated with keratoconjunctival disorders by strongly inhibiting the degradation of keratoconjunctival collagen.

Drawings

Fig. 1 is a graph showing the relationship between the concentration (nM) of RAR γ agonist a (R667) and collagen degradation (amount of hydroxyproline per well (μ g)). The vertical axis represents the value (%) when the amount of control hydroxyproline was set to 100.

Fig. 2 is a graph showing the relationship between the concentration (nM) of RAR γ agonist a (R667) and collagen contraction (collagen gel diameter (mm) in dish) when subconjunctival fibroblasts were used. Where "major" indicates that there is a statistically significant difference (p < 0.05).

Fig. 3 is a graph showing the relationship between the concentration (μ M) of RAR γ agonist a (R667) and collagen contraction (collagen gel diameter (mm) in dish) when corneal cells were used. Where "major" indicates that there is a statistically significant difference (p < 0.05).

Fig. 4 is a graph showing the relationship of the concentration (nM) of RAR γ agonist a (R667), RAR γ agonist B (CD 437) and RAR γ agonist C (BMS 961) with respect to collagen degradation when corneal cells were used. The vertical axis represents the Ratio (Ratio) of the amount of hydroxyproline per well (μ g) when RAR γ agonist and stimulator were added with the amount of hydroxyproline per well (μ g) set to 1 when RAR γ agonist and stimulator were not added, where "&" indicates that there was a statistically significant difference (p < 0.05).

Fig. 5 shows the relationship between the concentration (μ M) of RAR γ agonist a (R667) and the expression and activation of Matrix Metalloproteinase (MMP) when corneal cells were used. The top row of FIG. 5A shows MMP-1 expression and activation, the bottom row of FIG. 5A shows MMP-3 expression and activation, and FIG. 5B shows MMP-2 and MMP-9 expression and activation.

Fig. 6 shows the results of observation when Lipopolysaccharide (LPS) and RAR γ agonist a (R667) were administered into the corneal stroma in one eye of male japanese white rabbits. The top row (vehicle) is the result of administering only a solution containing RAR γ agonist a (R667) of the present invention, while the bottom row (0.1% R667) is the result of administering a solution containing RAR γ agonist a (R667).

Detailed Description

The therapeutic agent for keratoconjunctival disease of the present invention is not particularly limited, and may be any therapeutic agent having an RAR γ agonist as an active ingredient. The RAR γ agonist of the present invention refers to a compound that can significantly promote activation of RAR γ receptor by binding to RAR γ receptor, as compared with RAR α receptor or RAR β receptor.

Examples of such RAR γ agonists include (E) -4- (2- {3- [ (1H-pyrazol-1-yl) methyl ] -5,5,8, 8-tetramethyl-5, 6,7, 8-tetrahydronaphthalen-2-yl } vinyl) benzoic acid (R667) represented by the following formula (I), 6- [3- (1-adamantyl) -4-hydroxyphenyl ] -2-naphthoic acid (CD 437) represented by the following formula (II), 3-fluoro-4- [ 2-hydroxy-2- (5,5,8, 8-tetramethyl-5, 6,7, 8-tetrahydronaphthalen-2-yl) acetylamino ] benzoic acid (BMS 961) represented by the following formula (III), and (2E) -3- (4-carboxyphenyl) -1 (BMS 961) represented by the following formula (IV) - (5,5,8, 8-tetramethyl-5, 6,7, 8-tetrahydronaphthalen-2-yl) -2-propen-1-one oxime (NRX 204647: hereinafter also referred to as "RAR γ agonist D"), 4- [7- (1-adamantyl) -6-hydroxynaphthalen-2-yl ] benzoic acid represented by the following formula (V) (CD 1530: hereinafter also referred to as "RAR γ agonist E"), esters of such compounds (RAR γ agonists A, B, C, D and E) and salts of such compounds (RAR γ agonists A, B, C, D and E). Preferred examples of RAR γ agonists include RAR γ agonist A, RAR γ agonist B, RAR γ agonist C, esters of such compounds (RAR γ agonists A, B and C), and salts of such compounds (RAR γ agonists A, B and C).

[ chemical formula 1]

[ chemical formula 2]

[ chemical formula 3]

[ chemical formula 4]

[ chemical formula 5]

。

Further, other embodiments of the present invention include a method of treating a keratoconjunctival disorder characterized by administering to a subject an RAR γ agonist of the present invention, an RAR γ agonist of the present invention for use as a therapeutic agent for a keratoconjunctival disorder, and use of an RAR γ agonist of the present invention for the manufacture of a therapeutic agent for a keratoconjunctival disorder.

RAR γ agonist a, an ester thereof, and a salt thereof, which is one of the active ingredients of the therapeutic agent for keratoconjunctival disease of the present invention, are known compounds described in patent document 2. They can be manufactured according to the method described in patent document 2, or purchased as commercially available products. Examples of commercially available products include the following product names: paroxetine manufactured by Shanghai Haoyuanan Chemex.

Among the active ingredients of the therapeutic agent for keratoconjunctival disease of the present invention, RAR γ agonist B is a known compound described in non-patent document 1 and patent document 4, RAR γ agonist C is a known compound described in patent document 4, and RAR γ agonists D and E are known compounds described in the following documents (Shimono k. et al, Nat med. 17 (4): 454-460 (2011)). The compound, an ester thereof or a salt thereof may be produced according to a conventional method or purchased as a commercially available product. Examples of commercially available products include the following product names: for RAR γ agonist B, CD437 (ab 141305) manufactured by Abcam, and CD437 manufactured by Tocris Bioscience; for RAR γ agonist C, BMS961 manufactured by Tocris Bioscience, and CD1530 manufactured by Santa Cruz biotechnology; for RAR γ agonist E, CD1530 manufactured by Tocris Bioscience.

The ester of the above RAR γ agonists A, B, C, D and E, which is an active ingredient of the therapeutic agent for keratoconjunctival disease of the present invention, is not particularly limited, and may be any ester that is converted into RAR γ agonist A, B, C, D or E in a reaction with an enzyme or the like under physiological conditions in vivo. Such esters include: esters produced by reaction with primary alcohols such as methanol, ethanol, propanol, hexanol or dodecanol; esters produced by reaction with secondary alcohols such as isopropanol, sec-butanol, or 1-ethylpropanol; esters formed by reaction with tertiary alcohols such as t-butanol or 1-methyl-1-ethylpropanol; and esters formed by reaction with aminoalcohols such as 2-aminoethanol.

The above ester can be produced by a known method from the above RAR γ agonist A, B, C, D, E or a synthetic intermediate thereof.

The salt of the above RAR γ agonist A, B, C, D and E, which is an active ingredient of the therapeutic agent for keratoconjunctival disease of the present invention, is not particularly limited, and may be any pharmaceutically acceptable salt. Such salts include (1) inorganic acid salts as acid addition salts such as hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate and phosphate salts; and organic acid salts such as acetate, trifluoroacetate, benzoate, oxalate, malonate, succinate, maleate, fumarate, tartrate, citrate, methanesulfonate, ethanesulfonate, trifluoromethanesulfonate, benzenesulfonate, p-toluenesulfonate, glutamate and aspartate, and (2) metal salts such as sodium salt, potassium salt, calcium salt and magnesium salt as basic salts; inorganic salts such as ammonium salts; and organic amine salts such as triethylamine salt and guanidine salt.

In the present invention, a keratoconjunctival disorder refers to a condition in which the cornea or conjunctiva is damaged due to various factors, such as abnormal tear fluid, metabolic abnormality, or external damage. Examples of keratoconjunctival disorders include corneal ulcers, corneal epithelial abrasion, keratitis, dry eye, conjunctivitis, chronic superficial keratitis, corneal erosion, persistent corneal disorders, superficial punctate keratopathy, corneal epithelial defects, conjunctival epithelial defects, keratoconjunctivitis sicca, superior limbic keratoconjunctivitis, filamentous keratoconjunctivitis, infectious keratitis, non-infectious keratitis, infectious conjunctivitis, and non-infectious conjunctivitis. Has excellent improvement effect on the inhibition effect of collagen degradation. Further, in the present invention, corneal scars (scarring on the cornea) and conjunctival scars (scarring on the conjunctiva) associated with keratoconjunctival disorders are also examples of keratoconjunctival disorders. Exerts an excellent improving effect on the inhibition of collagen contraction.

The therapeutic agent for keratoconjunctival disease of the present invention may be administered orally or parenterally (intravenous administration, intramuscular administration, intraperitoneal administration, transdermal administration, intratracheal administration, intradermal administration, or subcutaneous administration) in the following forms, which are manufactured by mixing in suitable pharmacologically acceptable additives: tablets, capsules, powders, syrups, granules, fine granules, pills, liquids, suspensions, emulsions, transdermal agents, suppositories, ointments (preferably ophthalmic ointments), lotions, inhalants or injections.

These formulations are manufactured by well-known methods through the use of additives such as excipients, lubricants, binders, disintegrants, emulsifiers, stabilizers, flavoring agents, or diluents.

Examples of the excipient include organic excipients and inorganic excipients. Examples of organic excipients include: sugar derivatives such as lactose, sucrose, glucose, mannitol and sorbitol; starch derivatives such as corn starch, potato starch, alpha starch and dextrin; cellulose derivatives such as crystalline cellulose; acacia gum; dextran; and amylopectin. Examples of inorganic excipients include: light anhydrous silicic acid; and sulfates such as calcium sulfate.

Examples of lubricants include: stearic acid; metal salts of stearic acid such as calcium stearate and magnesium stearate; talc; colloidal silicon dioxide; waxes such as beeswax and spermaceti; boric acid; adipic acid; sulfates such as sodium sulfate; ethylene glycol; fumaric acid; sodium benzoate; d, L-leucine, sodium lauryl sulfate; silicic acid such as silica and silicic acid hydrate; and starch derivatives among the above excipients.

Examples of the binder include hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyvinylpyrrolidone, polyethylene glycol, and the compounds shown in the above excipients.

Examples of the disintegrant include: cellulose derivatives such as hydroxypropyl cellulose having a low degree of substitution, carboxymethyl cellulose, calcium carboxymethyl cellulose, and internally crosslinked calcium carboxymethyl cellulose; cross-linked polyvinylpyrrolidone; and chemically modified starch or cellulose derivatives such as carboxymethyl starch and sodium carboxymethyl starch.

Examples of emulsifiers include: colloidal clays such as soap clays and magnesium aluminum silicate; anionic surfactants such as sodium lauryl sulfate; cationic surfactants such as benzalkonium chloride; and nonionic surfactants such as polyoxyethylene alkyl ethers, polyoxyethylene sorbitan fatty acid esters, and sucrose fatty acid esters.

Examples of the stabilizer include: parabens such as methyl paraben and propyl paraben; alcohols such as chlorobutanol, benzyl alcohol and phenethyl alcohol; benzalkonium chloride; phenols such as phenol and cresol; thimerosal; acetic anhydride; and sorbic acid.

Examples of flavoring agents include: sweeteners such as sodium saccharin and aspartame; acidulants such as citric, malic and tartaric acids; and flavorings such as lemon extract and orange extract.

Diluents are compounds which are generally used as diluents. Examples of diluents include lactose, mannitol, dextrose, sucrose, calcium sulfate, hydroxypropyl cellulose, microcrystalline cellulose, water, ethanol, polyethylene glycol, propylene glycol, glycerol, starch, polyvinylpyrrolidone, and mixtures thereof.

The keratoconjunctival disease therapeutic agent of the present invention includes those in the form of an instillate in addition to the above-described dosage forms. The agent can be formulated by a known method by appropriately blending it with an isotonic agent, a buffer, a pH adjusting agent, a solubilizing agent, a thickening agent, a stabilizer, a preservative (antibacterial agent) and the like as an additive. Further, a stable instillation agent can be obtained by preparing a drug suspension by adding a pH adjuster, a thickener, a dispersant, and the like.

Examples of isotonic agents include glycerol, propylene glycol, sodium chloride, potassium chloride, sorbitol and mannitol.

Examples of buffers include phosphoric acid, phosphate, citric acid, acetic acid, and epsilon-aminocaproic acid.

Examples of the pH adjusting agent include hydrochloric acid, citric acid, phosphoric acid, acetic acid, sodium hydroxide, potassium hydroxide, boric acid, borax, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium carbonate, and sodium hydrogen carbonate.

Examples of the solubilizer include polysorbate 80, polyoxyethylene hydrogenated castor oil 60 and polyethylene glycol 4000.

Examples of thickeners and dispersants include: cellulose polymers such as hydroxypropylmethylcellulose and hydroxypropylcellulose; polyvinyl alcohol; and polyvinylpyrrolidone. Further, examples of the stabilizer include edetic acid and sodium edetate.

Examples of preservatives (antiseptics) include sorbic acid, potassium sorbate, benzalkonium chloride, benzethonium chloride, methylparaben, propylparaben, and chlorobutanol, which are commonly used. These preservatives can also be used in combination.

The instillate can have any pH within an acceptable range for ophthalmic formulations, but the pH is desirably set to 4.0-8.5.

For ointments, preferably ophthalmic ointments, customary bases such as white petrolatum or liquid paraffin may be used for the preparation.

The dose of the therapeutic agent for keratoconjunctival disease of the present invention may be appropriately changed according to the dosage form, severity of symptoms of a patient to whom the agent is administered, age, weight, judgment of a doctor, and the like. For oral agents, 0.01-5000 mg, preferably 0.1-2500 mg and more preferably 0.5-1000 mg can be administered per day for adults in general in 1 or several administrations. For the instillation, these can be administered at a concentration of 0.000001 to 10% (W/V), preferably 0.00001 to 3% (W/V), and more preferably 0.0001 to 1% (W/V) of the active ingredient in 1 or several administrations per day. For ophthalmic ointments, these can be administered at a concentration of 0.00001-10% (W/W), preferably 0.0001-3% (W/W), and more preferably 0.001-1% (W/W) of the active ingredient in 1 or several administrations per day.

Hereinafter, the present invention is explained in more detail while providing examples (test examples and formulation examples). However, the scope of the present invention is not limited thereto.

(example 1)

(test example) test for inhibition of three-dimensional collagen gel degradation in normal rabbit primary corneal cells by RAR γ agonist a

Normal rabbit corneal cells were used to evaluate the inhibitory effect of test compounds on three-dimensional collagen gel degradation according to the method of Nishida et al (Investigative Ophthalmology & Visual Science 42: 1247-1253 (2001)).

Primary keratocytes harvested from normal rabbit eyeballs were grown to confluent state and detached from the culture slides with 0.05% trypsin-EDTA. After washing in serum-free medium (product No. 11095; Gibco), the cell number was counted. The obtained primary corneal cells were mixed with Type I collagen solution Cellmatrix Type I-A (product No. 637-00653; Nitta Gelatin Inc.) And reconstitution buffer (product number 635-; nitta Gelatin Inc.) and dispensed into 24-well plates to a final concentration of 1 × 10⁵Cells/well to prepare collagen gels.

After the gel was prepared, by mixing a dimethylsulfoxide solution containing RAR γ agonist A (R667) (concentration of R667: 0.1 nM, 1 nM, 10 nM, 100 nM or 1000 nM) or a RAR γ agonist-free dimethylsulfoxide solution as a control, and 10 ng (final concentration of 10 ng/ml) of IL-1 β (product No. 201-LB-005; R) as an agonist&D Systems) and 60 μ g (final concentration 60 μ g/ml) plasminogen (product number P9156; sigma) was added to MEM medium, and the medium was overlaid on the previously prepared collagen gel to start the culture (at 37 ℃ and 5% CO)₂Under the conditions of (a).

After 48 hours of incubation, the supernatant was ultrafiltered, 100 μ l concentrated hydrochloric acid was added and heated, and collagen was hydrolyzed. The reaction solution after hydrolysis was dried under a nitrogen atmosphere by using Dry Thermo Uni (DTU-2C, Taitec co. Ltd.) and an evaporation head (E1-20 Taitec co. Ltd.), and then dissolved in 500 μ l of ultra pure water. The amount of hydroxyproline in the solution, which is a collagen degradation product, was measured according to the method of Bergman et al (Analytical Chemistry 35 (12): 1961-1965 (1963)) to evaluate the effect of RAR γ agonist A on the inhibition of collagen degradation. The results are shown in FIG. 1.

In this test, RAR γ agonist a showed a dose-dependent effect of inhibiting keratoconjunctival collagen degradation.

(preparation examples)

(pharmaceutical preparation example 1) instillation agent

In 100 ml

RAR gamma agonist A100 mg

800 mg of sodium chloride

Appropriate amount of polysorbate 80

Appropriate amount of disodium hydrogen phosphate

Appropriate amount of sodium dihydrogen phosphate

Proper amount of sterilized purified water.

RAR γ agonist a and other components described above were added to sterile purified water. The solution was mixed well to prepare a drip. By varying the amount of RAR γ agonist A or the like added, instillations at concentrations of 0.05% (W/V), 0.3% (W/V), 0.5% (W/V), or 1% (W/V) can be prepared.

(pharmaceutical preparation example 2) ophthalmic ointment

In 100 g

RAR gamma agonist A0.3 g

Liquid Paraffin 10.0 g

Proper amount of white vaseline.

RAR γ agonist a was added to white petrolatum and liquid paraffin, which were melted uniformly. The mixture was mixed well and then gradually cooled to prepare an ophthalmic ointment. By changing the amount of RAR gamma agonist A or the like added, an ophthalmic ointment can be prepared at a concentration of 0.05% (W/W), 0.1% (W/W), 0.5% (W/W), or 1% (W/W).

(pharmaceutical preparation example 3) tablet

In 100 mg

RAR gamma agonist A1 mg

Lactose 66.4 mg

Corn starch 20 mg

Carboxymethyl cellulose calcium 6 mg

Hydroxypropyl cellulose 6 mg

Magnesium stearate 0.6 mg.

RAR γ agonist a, corn starch and lactose were mixed in a mixer. Calcium carboxymethylcellulose and hydroxypropylcellulose were added to the mixture for granulation. The particle size of the resulting granules was adjusted after drying. Magnesium stearate is added and mixed with the adjusted granules, and the mixture is prepared into tablets with a tablet press. Further, by changing the amount of RAR γ agonist A or the like added, a tablet having a content of 0.1 mg, 10 mg or 50 mg in 100 mg can be prepared.

Example 2

(test example) test for inhibition of three-dimensional collagen gel contraction in various primary cells derived from normal rabbits by RAR γ agonist a

Primary subconjunctival fibroblasts and primary corneal cells were used to evaluate the inhibitory effect of test compounds on three-dimensional collagen gel contraction according to the method of Nishida et al.

Primary subconjunctival fibroblasts were grown and detached from the culture slides as in example 1. After washing, a cell suspension was prepared. The resulting suspension (1.1X 10)⁷Cell/well MEM), type I collagen solution (5 mg/ml), 10 XMEM reconstitution buffer and water were mixed on ice at 0.2:7:1:1:1.8 (volume ratio). A petri dish coated with 1% BSA was inoculated with 0.5 ml of this mixture, and the culture was incubated at 37 ℃ for one hour to prepare a collagen gel.

Subsequently, 0.5 ml of each of serum-free media was added to the above gel, and an amount (1 ng/ml) of TGF-. beta.1 (R & D Systems) and RAR. gamma. agonists at concentrations of 1 nM, 10 nM and 100 nM, respectively, were added to the serum-free media. The incubation of the gel was continued at 37 ℃ together with the gel to which the reagent-free, serum-free medium was added. The gel diameter was measured from the time of 24 hours. The measurement results of the gel diameter after 48 hours are shown in fig. 2.

Gel diameter was measured for primary corneal cells in a similar manner to primary subconjunctival fibroblasts. The results are shown in fig. 3.

As can be seen from fig. 2 and 3, using subconjunctival fibroblasts or corneal cells, RAR γ agonist a can inhibit not only collagen degradation but also collagen gel contraction by TGF. This demonstrates that RAR γ agonists contribute to collagen turnover (turn over) and have the effect of inhibiting tissue remodeling, i.e., fibrosis or scarring, that occurs after inflammation, bleeding, infection, surgery or injury in ocular tissues.

Example 3

(test example) test for inhibition of three-dimensional collagen gel degradation in primary corneal cells derived from normal rabbits by RAR γ agonist A, B or C

Primary corneal cells were used to evaluate the inhibitory effect of the test compound on the degradation of three-dimensional collagen gel by the same method as in example 1, according to the method of Nishida et al.

Primary corneal cells were grown and detached from the culture slides as in example 1. After washing, a cell suspension was prepared. The resulting suspension (1.1X 10)⁷Cell/well MEM), type I collagen solution (5 mg/ml), 10 XMEM reconstitution buffer and water were mixed on ice at 0.2:7:1:1:1.8 (volume ratio). A petri dish coated with 1% BSA was inoculated with 0.5 ml of this mixture, and the culture was incubated at 37 ℃ for one hour to prepare a collagen gel.

After the gel was prepared, the gel was prepared by dissolving RAR γ agonist A (R667) in dimethylsulfoxide (1 nM), RAR γ agonist B (CD 437) in dimethylsulfoxide (1 nM, 10 nM), RAR γ agonist C (BMS 961) in dimethylsulfoxide (10 nM) or RAR γ agonist-free dimethylsulfoxide (BMS 961) as a control, and IL-1 β (product No. 201-LB-005; R) as an agonist in 10 ng (final concentration 10 ng/ml)&D Systems) and 60 μ g (final concentration 60 μ g/ml) plasminogen (product number P9156, Sigma Aldrich) were added to the MEM medium and the culture was started (at 37 ℃ and 5% CO) by superimposing the medium on the previously prepared collagen gel₂Under the conditions of (a).

After 48 hours of culture, collagen was hydrolyzed, and the amount of hydroxyproline, which is a collagen degradation product, was measured by the same method as in example 1 to evaluate the effect of each RAR γ agonist on inhibition of collagen degradation. The results are shown in fig. 4.

In this test, not only RAR γ agonist a, but also RAR γ agonists B and C were shown to inhibit keratoconjunctival collagen degradation.

Example 4

(test example) test for inhibiting the expression and activation of MMP-1, 2, 3 and 9 by RAR gamma agonist

Secretion or expression of proteases, i.e. Matrix Metalloproteinases (MMPs), is thought to be associated with the degradation of type I collagen. In this regard, inhibition of expression and activation of MMPs-1, 2, 3 and 9 by RAR γ agonists was investigated.

Primary corneal cells collected from normal rabbit eyeballs were cultured in serum-free MEM medium for 24 hours. Agonizing RAR gammaAgent (R667) (concentration of R667: 1X 10)^-6 µM、1 × 10^-5 µM、1 × 10^-4 µM、1 × 10^-3 µM、1 × 10^-2Mum) was added to the obtained culture solution, and pretreatment was performed for 12 hours. As a positive control, 10 nM dexamethasone (Dex), which is a synthetic steroid, was added, and pretreatment was performed similarly. Subsequently, IL-1. beta. (0.1 ng/ml) was added for stimulation, and the supernatant was collected after 24 hours. The collected culture solutions were analyzed by western blot analysis and gelatin zymogram as described below.

(Western blot analysis)

In SDS-PAGE using a 10% polyacrylamide gel, after the supernatant of the collected culture solution was developed, the separated protein was transferred to a nitrocellulose filter. Subsequently, non-specific sites on nitrocellulose filters were blocked and incubated with anti-human MMP-1 antibodies (R & D Systems) or anti-rabbit MMP-3 antibodies (Daiichi Fine Chemical co., Ltd) at 4 ℃ for 24 hours. The detection was performed using ECL ® reagents (GE Healthcare).

The results are shown in fig. 5A. The bands for inactive PRO-MMP-1, PRO-MMP-3 and active MMP-1, MMP-3 were attenuated, respectively, with the amount depending on the concentration of RAR γ agonist a. Thus, RAR gamma agonist A was shown to inhibit MMP-1 and MMP-3 expression and activation concentration-dependently.

(analysis of gelatin enzyme spectra)

After the supernatant of the collected culture solution was developed on SDS-PAGE using a 10% polyacrylamide gel containing 0.1% gelatin, it was incubated with a TBS solution containing 2.5% Triton X-100 at room temperature for one hour. The incubated gel was stained with Coomassie brilliant blue (Wako Pure Chemical Industries, Ltd) solution and destained with 5% methanol-7.5% acetic acid solution (Nacalai Tesque, Inc).

The results are shown in fig. 5B. The bands for inactive PRO-MMP-2, PRO-MMP-9 and active MMP-2, MMP-9 were attenuated, respectively, with the amount depending on the concentration of RAR γ agonist a. Thus, RAR gamma agonist A was shown to inhibit MMP-2 and MMP-9 expression and activation concentration-dependently.

Example 5

(test example) test for inhibiting corneal haze and ulceration with RAR γ agonist A

General anesthesia was applied to male japanese white rabbits (2.5-3.5 kg body weight, 27 rabbits) by intramuscular administration of a mixture solution of ketamine and xylazine. Subsequently, local anesthesia was performed by 0.4% oxybuprocaine hydrochloride instillation. In addition, 30 μ l of 1% lps (sigma aldrich) was injected into the corneal stroma of one eye, while the other eye was not.

By using a micropipette, 50 μ l of a 0.1% PBS/0.1% polysorbate 80 solution containing RAR γ agonist a (R667: 0.1%, 24 mM) was instilled twice to the LPS-injected rabbits four times a day thereafter until day 10 after the injection on the day of LPS injection. As a control, a 0.1% PBS/0.1% polysorbate 80 solution (vehicle) without RAR γ agonist a was similarly administered. Figure 6 shows representative examples of controls and situations where RAR γ agonist a is administered.

In fig. 6, corneal haze and ulceration were observed in the control (medium) shown in the top row. However, in the case shown in the bottom row where RAR γ agonist a (0.1% R667) was administered, corneal haze and ulceration were not observed. Thus, RAR γ agonist a was revealed to inhibit corneal haze and ulceration.

Industrial applicability

An RAR γ agonist which is an active ingredient of the therapeutic agent for keratoconjunctival disorders of the present invention is useful for preventing keratoconjunctival disorders such as corneal ulcer, corneal epithelial abrasion, keratitis, dry eye, conjunctivitis, chronic superficial keratitis, corneal erosion, persistent corneal disorders, superficial punctate keratopathy, corneal epithelial defects, conjunctival epithelial defects, keratoconjunctivitis sicca, superior limbic keratoconjunctivitis, filamentary keratoconjunctivitis, infectious keratitis, non-infectious keratitis, infectious conjunctivitis and non-infectious conjunctivitis, or as a therapeutic agent for keratoconjunctival disorders by strongly inhibiting collagen degradation. Further, the RAR γ agonist can also be used as a therapeutic agent for corneal scars or conjunctival scars associated with keratoconjunctival disorders by strongly inhibiting collagen contraction.

Claims

Use of an RAR γ agonist or a salt thereof for the manufacture of a medicament for preventing or treating corneal ulcer in a subject in need thereof, wherein the RAR γ agonist is (E) -4- (2- {3- [ (1H-pyrazol-1-yl) methyl ] -5,5,8, 8-tetramethyl-5, 6,7, 8-tetrahydronaphthalen-2-yl } vinyl) benzoic acid or a salt thereof.
Use of an RAR γ agonist or a salt thereof for the manufacture of a medicament for inhibiting corneal haze in a subject in need thereof, wherein the RAR γ agonist is (E) -4- (2- {3- [ (1H-pyrazol-1-yl) methyl ] -5,5,8, 8-tetramethyl-5, 6,7, 8-tetrahydronaphthalen-2-yl } ethenyl) benzoic acid or a salt thereof.
Use of an RAR γ agonist or a salt thereof in the manufacture of a medicament for inhibiting scarring in the eye of a subject having a keratoconjunctival disorder, wherein the RAR γ agonist is (E) -4- (2- {3- [ (1H-pyrazol-1-yl) methyl ] -5,5,8, 8-tetramethyl-5, 6,7, 8-tetrahydronaphthalen-2-yl } ethenyl) benzoic acid or a salt thereof.
4. The use of any one of claims 1-3, wherein the medicament is formulated for oral, parenteral, intravenous, intramuscular, intraperitoneal, transdermal, intratracheal, intradermal, subcutaneous administration, or administration by instillation.
5. The use according to any one of claims 1 to 3, wherein the medicament is in the form of a powder, syrup, granule, suspension, emulsion, liquid, ointment or instillation.
6. The use of claim 5, wherein the medicament is in the form of an ophthalmic ointment, cream or instillate.
7. The use of claim 6, wherein the medicament comprises a solubilizing agent.
8. Use according to claim 7, wherein the solubilising agent is selected from polysorbate 80, polyoxyethylene hydrogenated castor oil 60 and polyethylene glycol 4000.
9. The use according to claim 8, wherein the solubilizer is polysorbate 80.
10. The use of claim 6, wherein the RAR γ agonist or salt thereof is present in the ophthalmic ointment at a concentration of 0.0001% to 3% (w/v).
11. The use according to claim 10, wherein the RAR γ agonist or salt thereof is present in the ophthalmic ointment at a concentration of 0.001% to 1% (w/v).
12. The use of claim 6, wherein the RAR γ agonist or salt thereof is present in the instillation at a concentration of 0.00001% to 3% (w/v).
13. The use of claim 12, wherein the concentration of the RAR γ agonist or salt thereof in the instillation is 0.0001% to 1% (w/v).
14. The use of claim 13, wherein the concentration of the RAR γ agonist or salt thereof in the instillation is 0.3% (w/v), 0.5% (w/v), or 1% (w/v).
15. The use of any one of claims 1-3 and 6-14, wherein the medicament is formulated as one or more daily doses.
16. The use of any one of claims 1-3, 6-9, and 12-14, wherein the medicament is formulated for administration by instillation.